RESUMEN
Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion, and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, they suggest that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Cóclea/cirugía , Pérdida Auditiva Provocada por Ruido/cirugía , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Muerte Celular , Línea Celular , Cóclea/metabolismo , Cóclea/patología , Modelos Animales de Enfermedad , Femenino , Cobayas , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/patología , Células Laberínticas de Soporte/metabolismo , Masculino , Ratones , Neuronas/trasplante , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
ATP receptor agonists and antagonists alter cochlear mechanics as measured by changes in distortion product otoacoustic emissions (DPOAE). Some of the effects on DPOAEs are consistent with the hypothesis that ATP affects mechano-electrical transduction and the operating point of the outer hair cells (OHCs). This hypothesis was tested by monitoring the effect of ATP-gamma-S on the operating point of the OHCs. Guinea pigs anesthetized with urethane and with sectioned middle ear muscles were used. The cochlear microphonic (CM) was recorded differentially (scala vestibuli referenced to scala tympani) across the basal turn before and after perfusion (20 min) of the perilymph compartment with artificial perilymph (AP) and ATP-gamma-S dissolved in AP. The operating point was derived from the cochlear microphonics (CM) recorded in response low frequency (200 Hz) tones at high level (106, 112 and 118 dB SPL). The analysis procedure used a Boltzmann function to simulate the CM waveform and the Boltzmann parameters were adjusted to best-fit the calculated waveform to the CM. Compared to the initial perfusion with AP, ATP-gamma-S (333 microM) enhanced peak clipping of the positive peak of the CM (that occurs during organ of Corti displacements towards scala tympani), which was in keeping with ATP-induced displacement of the transducer towards scala tympani. CM waveform analysis quantified the degree of displacement and showed that the changes were consistent with the stimulus being centered on a different region of the transducer curve. The change of operating point meant that the stimulus was applied to a region of the transducer curve where there was greater saturation of the output on excursions towards scala tympani and less saturation towards scala vestibuli. A significant degree of recovery of the operating point was observed after washing with AP. Dose response curves generated by perfusing ATP-gamma-S (333 microM) in a cumulative manner yielded an EC(50) of 19.8 microM. The ATP antagonist PPADS (0.1 mM) failed to block the effect of ATP-gamma-S on operating point, suggesting the response was due to activation of metabotropic and not ionotropic ATP receptors. Multiple perfusions of AP had no significant effect (118 and 112 dB) or moved the operating point slightly (106 dB) in the direction opposite of ATP-gamma-S. Results are consistent with an ATP-gamma-S induced transducer change comparable to a static movement of the organ of Corti or reticular lamina towards scala tympani.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad/farmacología , Potenciales Microfónicos de la Cóclea/efectos de los fármacos , Células Ciliadas Auditivas Externas/efectos de los fármacos , Emisiones Otoacústicas Espontáneas/efectos de los fármacos , Rampa Timpánica/efectos de los fármacos , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Análisis de Varianza , Animales , Potenciales Microfónicos de la Cóclea/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Cobayas , Células Ciliadas Auditivas Externas/fisiología , Masculino , Emisiones Otoacústicas Espontáneas/fisiología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Salicilatos/farmacología , Rampa Timpánica/fisiologíaRESUMEN
Extracellular adenosine has been suggested as a modulator of cochlear function. To date the release of adenosine into the extracellular spaces of the cochlea has not been demonstrated. Therefore, experiments were designed to examine whether adenosine release into perilymph could be detected in response to depolarization by high potassium concentrations or in response to asphyxia. For this purpose, the perilymph compartment of guinea pigs was perfused with an artificial perilymph and the effluent assayed for ATP, ADP, AMP and adenosine. Results indicate that potassium induced a slight, significant increase and asphyxia induced a very large, significant increase in adenosine levels in perilymph effluent. No changes in the levels of the other compounds were measured. It is concluded that depolarization and asphyxia can induce the release of adenosine into perilymph.
Asunto(s)
Adenosina/análisis , Asfixia/metabolismo , Perilinfa/química , Adenosina Difosfato/análisis , Adenosina Monofosfato/análisis , Adenosina Trifosfato/análisis , Análisis de Varianza , Animales , Asfixia/fisiopatología , Femenino , Cobayas , Masculino , Neurotransmisores/metabolismo , Perilinfa/metabolismo , Potasio/metabolismoRESUMEN
Cochleae of guinea pigs were evaluated for the presence of the metabotropic receptor, P2Y4. Evidence is presented that P2Y4 protein is expressed in the guinea pig cochleae using Western blot analysis. A single protein band of 35 kDa was detected with P2Y4 receptor-specific antibody. The cellular distribution of P2Y4 purinoceptor protein was determined by immunohistochemistry of the whole organ of Corti. Immunoreactive staining for P2Y4 was seen in most cells of the organ of Corti. Staining of Hensen's cells and Deiters' cells, especially the outer Deiters' cells, was more intense than staining of the outer hair cells, inner hair cells, and pillar cells. Staining intensity was greatest at the basal turn and progressively decreased in the upper turns with the apex showing the weakest staining pattern. This is the first demonstration of a metabotropic P2Y receptor in the guinea pig organ of Corti.
Asunto(s)
Órgano Espiral/química , Receptores Purinérgicos P2/análisis , Adenosina Trifosfato/análisis , Animales , Western Blotting , Cobayas , Inmunohistoquímica , Neurotransmisores/análisis , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2RESUMEN
Thapsigargin, a drug that inhibits sarco-endoplasmic reticulum Ca(2+) ATPases (SERCAs), was infused into the perilymph compartment of the guinea pig cochlea in increasing concentrations (0.1-10 microM) while sound evoked cochlear potentials were monitored. Thapsigargin significantly suppressed the compound action potential of the auditory nerve, cochlear microphonics, and increased N(1) latency at low (56 dB SPL) and high intensity (92 dB SPL) levels of sound, suppressed low intensity sound evoked summating potential (SP) and greatly increased the magnitude of the high intensity sound evoked SP. At 10 microM, the drug suppressed the cubic distortion product otoacoustic emissions (2f(1)-f(2)=8 kHz, f(2)=12 kHz) evoked by both high and low intensity primaries (45, 60, 70 dB SPL). Thapsigargin (10 microM; 30 min) increased the endocochlear potential slightly (5 mV). In chronic animals, thapsigargin (10 microM; 60 min) destroyed many outer hair cells and some inner hair cells, especially in the basal turns. These effects are consistent with the hypothesis that the inhibition of the SERCAs affects the function of the cochlear amplifier and outer hair cells to a greater degree than it affects other functions of the cochlea.
Asunto(s)
Potenciales Microfónicos de la Cóclea/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Emisiones Otoacústicas Espontáneas/efectos de los fármacos , Tapsigargina/farmacología , Estimulación Acústica , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Cóclea/efectos de los fármacos , Cóclea/patología , Inhibidores Enzimáticos/envenenamiento , Femenino , Cobayas , Células Ciliadas Auditivas Externas/patología , Masculino , Distorsión de la Percepción , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/envenenamientoRESUMEN
The hypothesis that the release of Ca(2+) from ryanodine receptor activated Ca(2+) stores in vivo can affect the function of the cochlea was tested by examining the effects of caffeine (1-10 mM) and ryanodine (1-333 microM), two drugs that release Ca(2+) from these intracellular stores. The drugs were infused into the perilymph compartment of the guinea pig cochlea while sound (10 kHz) evoked cochlear potentials and distortion product otoacoustic emissions (DPOAEs; 2f(1)-f(2)=8 kHz, f(2)=12 kHz) were monitored. Caffeine significantly suppressed the compound action potential of the auditory nerve (CAP) at low intensity (56 dB SPL; 3.3 and 10 mM) and high intensity (92 dB SPL; 10 mM), increased N1 latency at high and low intensity (3 and 10 mM) and suppressed low intensity summating potential (SP; 10 mM) without an effect on high intensity SP. Ryanodine significantly suppressed the CAP at low intensity (100 and 333 microM) and at high intensity (333 microM), increased N1 latency at low intensity (33, 100 and 333 microM) and at high intensity (333 microM) and suppressed low intensity SP (100 and 333 microM) and increased high intensity SP (333 microM). The cochlear microphonic (CM) evoked by 10 kHz tone bursts was not affected by caffeine at high or low intensity, and ryanodine had no effect on it at low intensity but decreased it at high intensity (10, 33, 100 and 333 microM). In contrast, caffeine (10 mM) and ryanodine (33 and 100 microM) significantly increased CM evoked by l kHz tone bursts and recorded from the round window. Caffeine (10 mM) and ryanodine (100 microM) reversibly suppressed the cubic DPOAEs evoked by low intensity primaries. Overall, low intensity evoked responses were more sensitive and were suppressed to a greater extent by both drugs. This is consistent with the hypothesis that release of Ca(2+) from ryanodine receptor Ca(2+) stores, possibly in outer hair cells and supporting cells, affects the function of the cochlear amplifier.